Architecture-Based Programming of Polymeric Micelles to Undergo Sequential Mesophase Transitions

Di- and triblock amphiphiles can form different mesophases ranging from micelles to hydrogels depending on their chemical structures, hydrophilic to hydrophobic ratios, and their ratio in the mixture. In addition, their different architectures dictate their exchange rate between the assembled and unimer states and consequently affect their responsiveness toward enzymatic degradation. Here we report the utilization of the different reactivities of di- and triblock amphiphiles, having exactly the same hydrophilic to lipophilic balance, toward enzymatic degradation as a tool for programming formulations to undergo sequential enzymatically induced transitions from (i) micelles to (ii) hydrogel and finally to (iii) dissolved polymers. We show that the rate of transition between the mesophases can be programmed by changing the ratio of the amphiphiles in the formulation, and that the hydrogels can maintain encapsulated cargo, which was loaded into the micelles. The reported results demonstrate the ability of molecular architecture to serve as a tool for programming smart formulations to adopt different structures and functions.


Instrumentation
HPLC: All measurements were recorded on a Waters Alliance e2695 separations module equipped with a Waters 2998 photodiode array detector. All solvents were purchased from Bio-Lab Chemicals and were used as received. All solvents are HPLC grade. 1 H and 13 C-NMR: spectra were recorded on Bruker Avance III 400MHz/100MHz spectrometer. Chemical shifts are reported in ppm and referenced to the solvent. The molecular weights of the dendron-PEG-dendron tri-block copolymers were determined by comparison of the areas of the peaks corresponding to the PEG block (3.63 ppm) and the protons peaks of the dendrons.
GPC: All measurements were recorded on Viscotek GPC max by Malvern using refractive index detector and PEG standards (purchased from Sigma-Aldrich) were used for calibration. DMF (purchased from Sigma, HPLC grade) was used as the mobile phase. Columns (2 x PSS GRAM 1000Å) were used at a column temperature of 50°C.

DLS:
All measurements were recorded on a Corduan Technology VASCO γ particle size analyzer.
Fluorescence Spectra: All spectra were recorded on an Agilent Technologies Cary Eclipse Fluorescence Spectrometer using quartz cuvettes.
Confocal microscopy: All images were taken in Olympus IX83 FLUOVIEW TM FV3000 inverted confocal microscope.

Cy-5 labeled DBA (mPEG5k-Lys(Cy-5)-[dend-(hexanoate)4], compound 5):
100 mg (0.016 mmol) of compound (3) were dissolved in DCM (1 mL) and TFA (200 µL). The mixture was allowed to stir for 1 hour and the reaction was monitored via HPLC. Once the Boc deprotection was confirmed by HPLC, DIPEA was added to the reaction mixture until fumes from it stopped coming out. After that MeOH-based LH20 SEC column was done to get rid of TFA.

Cy-3 labeled TBA (bPEG10k-bis-Lys(Cy-3)-[dend-(hexanoate)4], compound 12):
100 mg (0.008 mmol) of compound (10) were dissolved in DCM (1 mL) and TFA (200 µL). The mixture was allowed to stir for 1 hour and the reaction was monitored via HPLC. Once the Boc deprotection was confirmed by HPLC, DIPEA was added to the reaction mixture until fumes from it stopped coming out. After that MeOH-based LH20 SEC column was done to get rid of TFA.

General sample preparation:
A micellar solution (1:1 DBA: TBA) was prepared by mixing 5mg of each DBA (compound 3) and TBA (compound 10) in 1mL PBS giving a total polymers concentration of 10 mg/mL. Similarly, for the 2:1 DBA: TBA, 10mg of DBA (compound 3) and 5mg of TBA (compound 10) in 1 mL PBS give a total concentration of 15mg/mL. Vials were vortexed until full solubility was obtained, and then the solutions were sonicated for 15 minutes and filtered through a 0.22 µm nylon syringe filter. Measurements were performed at t=0 before the addition of the PLE enzyme.

General sample preparation:
A micellar solution of DBA was prepared by mixing 10mg of DBA (compound 3) in 1mL PBS to give a polymer concentration of 10mg/mL. This solution was then furhter diluted in PBS to prepare another solution of concentration 5mg/mL.
A micellar solution (1:1 DBA: TBA) was prepared by mixing 5mg of each DBA (compound 3) and TBA (compound 10) in 1mL PBS giving a total polymers concentration of 10 mg/mL. Similarly, for the 2:1 DBA: TBA, 10mg of DBA (compound 3) and 5mg of TBA (compound 10) in 1 mL PBS give a total concentration of 15mg/mL. Vials were vortexed until full solubility was obtained, and then the solutions were sonicated for 15 minutes and filtered through a 0.22 µm nylon syringe filter. Measurements were performed at t=0 before the addition of PLE enzyme.

Enzymatic degradation experiments of mixed micellar formulations:
A micellar solution (1:1 DBA: TBA) was prepared by mixing 5mg of each DBA (compound 3) and TBA (compound 10) in 1mL PBS giving a total polymers concentration of 10 mg/mL. Similarly, for the 2:1 DBA: TBA, 10mg of DBA (compound 3) and 5mg of TBA (compound 10) in 1 mL PBS give a total concentration of 15mg/mL. Vials were vortexed until full solubility was obtained and then placed in an ultrasonic bath for 15 minutes. PLE was added to yield a final concentration of 0.36 µM and degradation was followed at 37°C by monitoring the area under the peak of the parent amphiphile and hydrolyzed polymer by HPLC at 297 nm. Each experiment was conducted thrice; the reported values in each time point are the mean value, and the standard deviation is the error (results shown in main text, Figures 1 and 4).
To monitor the thermodynamic stability of the mixed micelles obtained from mixing DBA and TBA in 1:1 and 2:1, control experiments were done without adding enzyme. The degradation was followed at 37°C by monitoring the area under the peak of the parent amphiphile by HPLC at 297 nm. Each experiment was conducted thrice; the reported values in each time point are the mean value, and the standard deviation is the error.

Enzymatic degradation experiments of non-mixed micellar formulations:
5mg/mL solution of DBA was prepared in PBS. Vials were vortexed until full solubility was obtained and then placed in an ultrasonic bath for 15 minutes. PLE was added to yield a final concentration of 0.36 µM and degradation was followed at 37°C by monitoring the area under the peak of the parent amphiphile and hydrolyzed polymer by HPLC at 297 nm. Each experiment was a. b.
a. b.
conducted thrice; the reported values in each time point are the mean value, and the standard deviation is the error. For DLS, the solution was filtered through a 0.22 µm nylon syringe filter and measurements were done before and after micellar disassembly.

Rheology measurements:
Rheological measurements were performed using a controlled-stress rheometer (AR-G2, TA instruments, USA). An 8 mm diameter flat-plate geometry with a rough surface was used for the b. a. study. The viscous elastic region was determined by strain sweep from 0.01 to 100% strain at 1Hz frequency at 25°C, with a gap size of 0.9 mm. Figure S20: Amplitude sweep tests of the hydrogels obtained from (a) 1:1 DBA (compound 3, 5 mg/mL) and TBA (compound 10, 5 mg/mL), (b) in 2:1 DBA (compound 3, 10 mg/mL) and TBA (compound 10, 5 mg/mL), and (c) only TBA (5 mg/mL) at a constant frequency of 1Hz.

HRSEM measurements:
All images were taken using a Zeiss Gemini 300 high resolution scanning electron microscope in high vacuum, WD~5mm, 3kV.

Analysis of the composition of the formed hydrogels:
The solution above the hydrogel was removed and the remaining hydrogel was washed 3 times with PBS and then dissolved in acetonitrile. The HPLC analysis shows the presence of 11% partly hydrolyzed amphiphiles, 12% DBA and 77% TBA. Figure S23: HPLC result after adding acetonitrile to the gel formed after the degradation of the diblock amphiphiles.

Minutes
TBA DBA Partly hydrolyzed amphiphiles

Fluorescence Measurements:
A micellar solution (1:1 DBA: TBA) was prepared by mixing 4.5 mg of each DBA (compound 3) and TBA (compound 10) and 0.5 mg of each compound: Cy-5 labeled DBA (5) and Cy-3 labeled TBA (12) in 1 mL PBS giving a total polymers concentration of 10mg/mL. The solution was vortexed until full solubility was obtained and then placed in an ultrasonic bath for 15 minutes. PLE was added to yield a final concentration of 0.36 µM and fluorescence was measured at 37°C by exciting at 512 nm (Cy-3 excitation), shown in main text, Figure 2a. Figure S24: Fluorescence spectrum of micelles containing both dyes at t=0 and t=24h in the absence of PLE.

Hydrogel Degradation:
To study the stability of hydrogel formed from the enzymatic degradation experiment (Section 5.1) we added BSA and an excess of an enzyme. The solution above the hydrogel was removed and the remaining hydrogel was washed 3 times with PBS. Two parallel experiments were conducted, first, 500µL of 3.5mg/mL of BSA in PBS was added and second, 500µL of 3.5mg/mL of BSA along with 1µM of PLE in PBS was added.

Nile Red Encapsulation:
A micellar solution (1:1 DBA: TBA) was prepared by mixing 5 mg of each DBA (compound 3) and TBA (compound 10) and Nile red in 1 mL PBS giving a total polymeric concentration of 10 mg/mL and 10 µM of Nile red. The sample was vortexed until full solubility was obtained and then placed in an ultrasonic bath for 15 minutes. PLE was added to yield a final concentration of 0.36 µM and degradation was followed at 37°C by measuring the fluorescence by exciting at 500 nm. Figure S25: Gel formed from TBA (compound 10) in water with a concentration of 5mg/0.5mL using the thin-film hydration in the left vial, and 5mg/0.25mL using solvent exchange method in the right vial (ethanol was used as a solvent).

Gel formed from the tri-block copolymer after the enzymatic degradation
In the first experiment DBA (compound 3, 5mg/mL) and TBA (compound 10, 5mg/mL) were mixed in a 1:1 ratio with a total final concentration of 10 mg/mL, while in the second experiment DBA (compound 3, 10mg/mL) and TBA (compound 10, 5mg/mL) were mixed in a 2:1 ratio with a total final concentration of 15 mg/mL, the enzyme (PLE) concentration was 0.36 µM.